This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The flavoenzyme UDP-galactopyranose mutase (UGM) catalyzes the interconversion of UDP-galactopyranose and UDP-galactofuranose. Galactofuranose residues, which are not found in mammalian cells, are then incorporated into the cell walls of certain bacteria, including Mycobacterium tuberculosis, making UGM an attractive drug target. UGM is a flavoenzyme, but the precise role of the cofactor has been unclear due to the lack of reduction/oxidation chemistry in this isomerization. We propose that turnover occurs by a covalent flavin-galactose iminium species. Using NaCNBH3 to trap this adduct, analysis of the resulting species by LCMS shows a peak corresponding to the molecular weight of the proposed alkylated flavin, as well as an absorbance spectrum indicative of an N(5) alkylflavin. In order to investigate how the enzyme allows for this unique chemistry, we have begun to mutate conserved residues around the active site in order to establish the mechanistic roles of certain amino acids. A protocol for removal of the noncovalently bound flavin and replacement with isotopically labeled flavin has been developed to allow for NMR studies of the flavin binding site, as well as to use NMR to visualize the connectivities of the covalent alkylflavin.